Obligate domain-swapped dimer of cyanovirin with enhanced anti-viral activity

ABSTRACT

A purified or isolated obligate domain-swapped dimer of CVN; an isolated or purified nucleic acid encoding at least one obligate domain-swapped dimer of CVN, optionally in the form of a vector; a host cell comprising such an isolated or purified nucleic acid; a composition comprising (i) the obligate domain-swapped dimer of CVN or the isolated or purified nucleic acid encoding at least one obligate domain-swapped dimer of CVN, optionally in the form of a vector, and (ii) a carrier therefor; a method of inhibiting a viral infection of a mammal, which method comprises administering to the mammal an antiviral effective amount of the aforementioned composition, whereupon the viral infection of the mammal is inhibited; and a method of making an obligate domain-swapped dimer of CVN, which method comprises introducing at least one mutation in the linker region of wild-type CVN, whereupon an obligate domain-swapped dimer of CVN is obtained.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an obligate domain-swapped dimer ofcyanovirin (CVN); a nucleic acid encoding same, optionally in the formof a vector; a host cell comprising such a nucleic acid; a compositioncomprising (i) the obligate domain-swapped dimer of CVN or the nucleicacid encoding same, optionally in the form of a vector, and (ii) acarrier therefor; a method of inhibiting a viral infection of a mammalby administering to the mammal an antiviral effective amount of theaforementioned composition; and a method of making an obligatedomain-swapped dimer of CVN by introducing at least one mutation in thelinker region of wild-type CVN.

BACKGROUND OF THE INVENTION

CVN, a protein isolated from cyanobacteria, is known to have anti-viralactivity. By interfering with the interaction of the viral envelopeglycoprotein gp120 of human immunodeficiency viruses (HIV) with thesurface of a cell, CVN can block the entry of HIV into the cell atnanomolar concentrations (Boyd et al., Animicrob. Agents Chemother. 41:1521-1530(1997); Dey et al., J. Virol. 74: 4562-4569 (2000)). CVN:gp120interactions are governed by high affinity binding of CVN to the D1 andD3 arms of oligomannose-8 and oligomannose-9, two oligosaccharides thatare abundant on the surface of HIV (Bewley et al., J. Am. Chem. Soc.123: 3892-3902 (2001)). This unprecedented specificity arises from thepresence of two extensive carbohydrate binding pockets that are specificfor the disaccharide oligomannose-α (1-2) oligomannose-α, i.e., thetermini of the more accessible D1 and D3 arms of oligomannose-8 andoligomannose-9 (Bewley, Structure 9: 931-940 (2001)).

It is an object of the present invention to enhance the anti-viralactivity of CVN. This and other objects and advantages of the presentinvention, as well as additional inventive features, will becomeapparent from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a purified or isolated obligatedomain-swapped dimer of CVN. Also provided are an isolated or purifiednucleic acid encoding at least one obligate domain-swapped dimer of CVN,optionally in the form of a vector, and a host cell comprising such anisolated or purified nucleic acid. A composition comprising (i) theobligate domain-swapped dimer of CVN or the isolated or purified nucleicacid encoding at least one obligate domain-swapped dimer of CVN,optionally in the form of a vector, and (ii) a carrier therefor is alsoprovided.

Accordingly, a method of inhibiting a viral infection of a mammal isalso provided. The method comprises administering to the mammal anantiviral effective amount of the aforementioned composition, whereuponthe viral infection of the mammal is inhibited.

A method of making an obligate domain-swapped dimer of CVN is alsoprovided. The method comprises introducing at least one mutation in thelinker region of wild-type CVN, whereupon an obligate domain-swappeddimer of CVN is obtained.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, at least in part, on the discoverythat an obligate domain-swapped dimer of CVN has enhanced antiviralactivity. Accordingly, the present invention provides a purified orisolated obligate domain-swapped dimer of cyanovirin. By “cyanovirin” ismeant the isolated and purified native antiviral protein referred to ascyanovirin-N and obtained from Nostoc ellipsosporum as well as anyrelated, functionally equivalent protein, peptide or derivative thereof.Desirably, the overall length of the linker region of CVN and theorientation of the two domains of CVN relative to each other arechanged, such that the torsion angles are changed and a rigid structureis imposed. Preferably, the dimer is a tetravalent carbohydrate bindingprotein, which preferably is stable at a pH from about 2.3 to about 8.0.Preferably, the dimer comprises the amino acid sequence of wild-type CVNin which there is at least one mutation in the linker region.Preferably, the at least one mutation is in the region from about aminoacid position 48 to about amino acid position 54. In a preferredembodiment of the dimer, the glutamine at amino acid position 50 of CVNis deleted. Alternatively, a proline is inserted in the region fromabout amino acid position 50 to about amino acid position 53 or an aminoacid in the region from about amino acid position 50 to about amino acidposition 53 is substituted with a proline.

Cyanovirin (CVN) can be isolated from N. ellipsosporum in accordancewith methods known in the art. See, e.g., U.S. Pat. No. 5,962,653.Alternately, the polypeptide can be synthesized using standard peptidesynthesizing techniques well-known to those of skill in the art (e.g.,as summarized in Bodanszky, Principles of Peptide Synthesis(Springer-Verlag, Heidelberg: 1984)). In particular, the polypeptide canbe synthesized using the procedure of solid-phase synthesis (see, e.g.,Merrifield, J. Am. Chem. Soc. 85: 2149-54 (1963); Barany et al., Int. J.Peptide Protein Res. 30: 705-739 (1987); and U.S. Pat. No. 5,424,398).If desired, this can be done using an automated peptide synthesizer.Removal of the t-butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups andseparation of the polypeptide from the resin can be accomplished by, forexample, acid treatment at reduced temperature. Thepolypeptide-containing mixture then can be extracted, for instance, withdimethyl ether, to remove non-peptidic organic compounds, and thesynthesized polypeptide can be extracted from the resin powder (e.g.,with about 25% w/v acetic acid). Following the synthesis of thepolypeptide, further purification (e.g., using high performance liquidchromatography (HPLC)) optionally can be done in order to eliminate anyincomplete polypeptides or free amino acids. Amino acid and/or HPLCanalysis can be performed on the synthesized polypeptide to validate itsidentity.

Since the nucleotide and corresponding amino acid sequences ofcyanovirin are known (see, e.g., SEQ ID NOS: 1 and 2, respectively, inU.S. Pat. No. 5,843,882), cyanovirin also can be recombinantly producedor synthesized in accordance with methods known in the art. See, e.g.,U.S. Pat. Nos. 5,821,081 and 5,843,882 and the references cited under“Examples.”

An obligate domain-swapped dimer of CVN can be made in accordance withmethods known in the art. In this regard, mutations, such as insertions,deletions, substitutions and/or inversions, can be introduced in thelinker region at the amino acid level or at the nucleic acid level. Forinstance, site-specific mutations can be introduced by ligating into anexpression vector a synthesized oligonucleotide comprising the modifiedsite. Alternately, oligonucleotide-directed site-specific mutagenesisprocedures can be used, such as disclosed in Walder et al., Gene 42: 133(1986); Bauer et al., Gene 37: 73 (1985); Craik, Biotechniques: 12-19(January 1995); U.S. Pat. Nos. 4,518,584 and 4,737,462; Carter et al.,Nucl. Acids Res. 13: 4331 (1986); and Zoller et al., Nucl. Acids Res.10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene 34: 315(1985)), restriction selection mutagenesis (Wells et al., Philos. Trans.R. Soc. London SerA 317: 415 (1986)) and DNA synthesis of the mutatedCVN. A preferred means for introducing mutations is the QuikChangeSite-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.). Whenmodifying the nucleic acid so that a new amino acid is substituted forthat which is naturally occurring, the codon encoding the amino acidsequence to be substituted may be any of the alternative codons known tocode for the particular amino acid (see, e.g. Lewin GENES V OxfordUniversity Press, page 172 (1994)).

The dimer can be optionally glycosylated, amidated, carboxylated,phosphorylated, esterified, N-acylated or converted into an acidaddition salt. Methods of protein modification (e.g., glycosylation,amidation, carboxylation, phosphorylation, esterification, N-acylation,and conversion into acid addition salts) are known in the art.Preferably, the dimer is not glycosylated.

The anti-viral, e.g., anti-HIV, activity of the dimer can be assessed inaccordance with the assay set forth in Example 2 or in a series ofinterrelated in vitro antiviral assays (Gulakowski et al., J. Virol.Methods 33: 87-100 (1991)), which accurately predict for antiviralactivity in humans. These assays measure the ability of compounds toprevent the replication of HIV and/or the cytopathic effects of HIV onhuman target cells. These measurements directly correlate with thepathogenesis of HIV-induced disease in vivo.

Fusion proteins comprising at least one dimer and conjugates comprisingat least one dimer also can be generated. Such fusion proteins andconjugates can be generated in accordance with the methods of U.S. Pat.Nos. 5,962,688 and 6,245,737 as well as International Patent ApplicationNo. WO 00/53213.

An isolated or purified nucleic acid encoding at least one obligatedomain-swapped dimer of CVN, optionally in the form of a vector, is alsoprovided. The nucleic acid molecule can be cloned into any suitablevector and can be used to transform or transfect any suitable host. Theselection of vectors and methods to construct them are commonly known topersons of ordinary skill in the art and are described in generaltechnical references (see, in general, “Recombinant DNA Part D,” Methodsin Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)and the references cited herein under “Examples”). Desirably, the vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant or animal) into which the vector isto be introduced, as appropriate and taking into consideration whetherthe vector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host. In this regard, the vector can encode two, three,four, five, six, seven and even eight of the obligate domain-swappeddimers of CVN, which can be the same or different.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid sequence as described above or a portionthereof ligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColE 1, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

Suitable vectors include those designed for propagation and expansion orfor expression or both. A preferred cloning vector is selected from thegroup consisting of the pUC series, the pBluescript series (Stratagene,LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEXseries (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNMI 149, also can be used.Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech).

An expression vector can comprise a native or normative promoteroperably linked to an isolated or purified nucleic acid molecule asdescribed above. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theskill in the art. Similarly, the combining of a nucleic acid molecule asdescribed above with a promoter is also within the skill in the art.

Optionally, the isolated or purified nucleic acid molecule, upon linkagewith another nucleic acid molecule, can encode a fusion protein. Thegeneration of fusion proteins is within the ordinary skill in the art(see, e.g., references cited under “Example”) and can involve the use ofrestriction enzyme or recombinational cloning techniques (see, e.g.,Gateway™ (Invitrogen, Carlsbad, Calif.)). See, also, U.S. Pat. Nos.5,314,995 and 5,962,688 and International Patent Application No. WO00/53213.

Also in view of the above, the present invention provides a host cellcomprising and expressing an above-described isolated or purifiednucleic acid molecule, optionally in the form of a vector, as describedabove. Examples of host cells include, but are not limited to, bacteria,such as species within the genera Escherichia (e.g., E. coli TB-1, TG-2,DH5α, XL-Blue MRF′ (Stratagene), SA2821 and Y1090), Bacillus (e.g., B.subtilis), Pseudomonas (e.g., P. aerugenosa), and Salmonella, as well aslactobacilli, yeast, such as S. cerevisiae or N. crassa, insect cells(e.g., Sf9, Ea4 and baculoviral systems (e.g., as described by Luckow etal., Bio/Technology 6: 47 (1988)), mammalian cells, including humancells, and established cell lines, such as the COS-7, C127, 3T3, CHO,HeLa, and BHK cell lines, and the like. The ordinarily skilled artisanis, of course, aware that the choice of expression host hasramifications for the type of polypeptide produced. For instance, theglycosylation of polypeptides produced in yeast or mammalian cells(e.g., COS-7 cells) will differ from that of polypeptides produced inbacterial cells, such as E. coli.

The present invention also provides a composition comprising (i) eitherof an above-described dimer or nucleic acid, optionally in the form of avector, and (ii) a carrier therefore. Suitable carriers, such aspharmaceutically acceptable carriers, are well-known in the art, and arereadily available. The choice of carrier will be determined in part bythe particular route of administration and whether a nucleic acidmolecule or dimer is being administered. Accordingly, there is a widevariety of suitable formulations for use in the context of the presentinvention, and the present invention expressly provides a pharmaceuticalcomposition that comprises an active agent of the invention and apharmaceutically acceptable carrier therefor. The following methods andcarriers are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the active agent dissolved indiluent, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth. Pastilles can comprise the active ingredientin an inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients/carriers as are known in the art.

An active agent of the present invention, either alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also canbe formulated as pharmaceuticals for non-pressured preparations such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, active agents of the present invention can be made intosuppositories by mixing with a variety of bases, such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration can be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.Further suitable formulations are found in Remington's PharmaceuticalSciences, 17th ed., (Mack Publishing Company, Philadelphia, Pa.: 1985),and methods of drug delivery are reviewed in, for example, Langer,Science 249: 1527-1533 (1990). Similarly, the active ingredient can becombined with a lubricant as a coating on a condom. Indeed, preferably,the active ingredient is applied to any contraceptive device, including,but not limited to, a condom, a diaphragm, a cervical cap, a vaginalring, and a sponge. Such formulations allow for vaginal, rectal, penileor other topical routes of administration in the inhibition of viralinfection through sexual activity. In this regard, lactobacilli, whichexpress the dimer, can be introduced into the vagina.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

In the event that it becomes desirable or necessary to enhance furtherthe stability of the dimer, such methods are known in the art. See,e.g., International Patent Application No. WO 00/53213.

The above compositions can contain other active agents, such as thosewhich inhibit viral infection. Representative examples of theseadditional active agents include antiviral compounds, virucides,immunomodulators, immunostimulants, antibiotics and absorptionenhancers. Exemplary antiviral compounds include AZT, ddI, ddC,gancylclovir, fluorinated dideoxynucleosides, normucleoside analogcompounds, such as nevirapine (Shih et al., PNAS 88: 9878-9882 (1991)),TIBO derivatives, such as R82913 (White et al., Antiviral Res. 16:257-266 (1991)), BI-RJ-70 (Merigan, Am. J. Med. 90 (Suppl.4A): 8S-17S(1991)), michellamines (Boyd et al., J. Med. Chem. 37: 1740-1745 (1994))and calanolides (Kashman et al., J. Med. Chem. 35: 2735-2743 (1992)),nonoxynol-9, gossypol and derivatives, and gramicidin (Bourinbair etal., Life Sci./Pharmocol. Lett. 54: PL5-9 (1994); and Bourinbair et al.,Contraception 49: 131-137 (1994)). Exemplary immunomodulators andimmunostimulants include various interleukins, sCD4, cytokines, antibodypreparations, blood transfusions, and cell transfusions. Exemplaryantibiotics include antifingal agents, antibacterial agents, andanti-Pneumocystilis carnii agents. Exemplary absorption enhancersinclude bile salts and other surfactants, saponins, cyclodextrins, andphospholipids (Davis, Pharm. Pharmacol. 44 (Suppl. 1): 186-190 (1992)).

In view of the above, the present invention also provides a method ofinhibiting a viral infection in a mammal. The method comprisesadministering to the mammal an antiviral effective amount of anabove-described dimer, nucleic acid, optionally in the form of a vector,or composition comprising same, whereupon the viral infection of themammal is inhibited. Preferably, the mammal is a human and the viralinfection is human immunodeficiency viral infection.

The dimers can be used to inhibit a virus, specifically a retrovirus,more specifically an immunodeficiency virus, such as the humanimmunodeficiency virus, i.e., HIV-1 or HIV-2. The dimers also can beused to inhibit other retroviruses as well as other viruses. Examples ofviruses that may be inhibited in accordance with the present inventioninclude, but are not limited to, Type C and Type D retroviruses, HTLV-1,HTLV-2, HIV, FIV, FLV, SIV, MLV, BLV, BIV, equine infectious virus,anemia virus, avian sarcoma viruses, such as Rous sarcoma virus (RSV),hepatitis type A, B, non-A and non-B viruses, arboviruses, varicellaviruses, human herpes virus (e.g., HHV-6), measles, mumps, influenza,and rubella viruses.

Generally, when an above-described dimer is administered to an animal,such as a mammal, in particular a human, such as in accordance with anyof the methods set forth in International Patent Application No. WO00/53213, it is preferable that the dimer is administered in a dose offrom about 1 to about 1,000 micrograms of the dimer per kg of the bodyweight of the host per day when given parenterally. However, this dosagerange is merely preferred, and higher or lower doses can be chosen inappropriate circumstances. For instance, the actual dose and schedulecan vary depending on whether the composition is administered incombination with other pharmaceutical compositions, or depending oninterindividual differences in pharmacokinetics, drug disposition, andmetabolism. One skilled in the art easily can make any necessaryadjustments in accordance with the necessities of the particularsituation.

Those of ordinary skill in the art can easily make a determination ofthe amount of an above-described isolated and purified nucleic acidmolecule, which is optionally in the form of a vector, to beadministered to an animal, such as a mammal, in particular a human. Thedosage will depend upon the particular method of administration,including any vector or promoter utilized. For purposes of consideringthe dose in terms of particle units (pu), also referred to as viralparticles, it can be assumed that there are 100 particles/pfu (e.g.,1×1012 pfu is equivalent to 1×1014 pu). An amount of recombinant virus,recombinant DNA vector or RNA genome sufficient to achieve a tissueconcentration of about 102 to about 1012 particles per ml is preferred,especially of about 106 to about 1010 particles per ml. In certainapplications, multiple daily doses are preferred. Moreover, the numberof doses will vary, depending on the means of delivery and theparticular vector administered.

Administration of a dimer with other anti-retroviral agents andparticularly with known RT inhibitors, such as ddC, AZT, ddI, ddA, orother inhibitors that act against other HIV proteins, such as anti-TATagents, is expected to inhibit most or all replicative stages of theviral life cycle. The dosages of ddC and AZT used in AIDS or ARCpatients have been published. A virustatic range of ddC is generallybetween 0.05 μM to 1.0 μM. A range of about 0.005-0.25 mg/kg body weightis virustatic in most patients. The preliminary dose ranges for oraladministration are somewhat broader, for example 0.001 to 0.25 mg/kggiven in one or more doses at intervals of 2, 4, 6, 8, 12; etc. hours.Currently, 0.01 mg/kg body weight ddC given every 8 hrs is preferred.When given in combined therapy, the other antiviral compound, forexample, can be given at the same time as the dimer or the dosing can bestaggered as desired. The two drugs also can be combined in acomposition. Doses of each can be less when used in combination thanwhen either is used alone.

It will also be appreciated by one skilled in the art that a DNAsequence of a cyanovirin or conjugate thereof of the present inventioncan be inserted ex vivo into mammalian cells previously removed from agiven animal, in particular a human, host. Such cells can be employed toexpress the dimer in vivo after reintroduction into the host.Feasibility of such a therapeutic strategy to deliver a therapeuticamount of an agent in close proximity to the desired target cells andpathogens, i.e., virus, more particularly retrovirus, specifically HIVand its envelope glycoprotein gp120, has been demonstrated in studieswith cells engineered ex vivo to express sCD4 (Morgan et al., AIDS Res.Hum. Retrovir. 10: 1507-1515 (1994)). It is also possible that, as analternative to ex vivo insertion of the DNA sequences of the presentinvention, such sequences can be inserted into cells directly in vivo,such as by use of an appropriate viral vector. Such cells transfected invivo are expected to produce viral-inhibiting amounts of dimer directlyin vivo.

Given the present disclosure, it will be additionally appreciated that anucleic acid encoding a dimer can be inserted into suitable nonmammalianhost cells, and that such host cells will express the dimer directly invivo within a desired body compartment of an animal, in particular ahuman, in an amount sufficient to inhibit viral infection.

In a preferred embodiment of the present invention, a method offemale-controllable prophylaxis against viral infection comprises theintravaginal administration and/or establishment of, in a female human,a persistent intravaginal population of lactobacilli that have beentransformed with a dimer-encoding sequence to produce, over a prolongedtime, levels of the dimer, directly on or within the vaginal and/orcervical and/or uterine mucosa, sufficient to inhibit viral infection.

The dimer, whether in the form of a protein or nucleic acid encodingsame as described above, can be used in other methods. See, e.g., U.S.Pat. No. 6,015,876 and International Patent Application No. WO 00/53213.The dimer (or conjugate or fusion protein thereof) also can be used toremove virus from a sample as described in WO 00/53213.

A method of making an obligate domain-swapped dimer of CVN is alsoprovided. The method comprises introducing at least one mutation in thelinker region of wild-type CVN, whereupon an obligate domain-swappeddimer of CVN is obtained. The mutations can be introduced at the nucleicacid or amino acid level in accordance with methods known in the art asdiscussed above. Preferably, the at least one mutation is in the regionfrom about amino acid position 48 to about amino acid position 54.Preferably, glutamine at amino acid position 50 is deleted.Alternatively, a proline is inserted in the region from about amino acidposition 50 to about amino acid position 53 or an amino acid in theregion from about amino acid position 50 to about amino acid position 53is substituted with a proline.

EXAMPLES

The following examples serve to illustrate the present invention and arenot intended to limit its scope in any way.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1997),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1998),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y. (1988),-   Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), and-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd    edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y. (1989).

Nuclear magnetic resonance (NMR) spectra were recorded on a BrukerDMX500 MHz spectrometer equipped with x,y,z-shielded gradient tripleresonance probes. Analytical ultracentrifugation experiments wereconducted at 20.0° C. on a Beckman Optima XL-A analyticalultracentrifuge. Protein samples were prepared in 20 mM sodium phosphatebuffer, pH 6.4, and loaded into the ultracentrifuge cells at loadingconcentrations of 20 μM. Data were analyzed in terms of a single idealsolute to obtain the buoyant molecular mass, M(1-νρ), using the OptimaXL-A data analysis software (Beckman). The value for the experimentalmolecular mass, M, was determined using calculated values for thedensity, ρ (determined at 20.0° C. using standard tables), and partialspecific volume, ν (calculated on the basis of amino acid composition).Site-directed mutagenesis on a pet46A plasmid containing a syntheticgene encoding wild-type CVN was performed using the Stratagene (LaJolla, Calif.) QuikChange™ Kit according to the manufacturer'sinstructions. Forward and reverse primers with respective sequences of5′-GACGGTTCCCTGAAATGGCCGTCCAACTTCATCGAAACC-3′ [SEQ ID NO: 1] and5′-GGTTTCGATGAAGTTGGACGGCCATTTCAGGGAACCGTC-3′ [SEQ ID NO: 2] were usedfor the PCR reactions, and were gel-purified before use (Lofstrand Labs,Gaithersburg, Md.). DNA sequencing of the deletion mutant insert wasperformed on an ABI PRISM DNA sequencer (Applied Biosystems) onpolymerase chain reaction (PCR) products generated with RhodamineTerminator (Perkin Elmer) labeling of the sequence between the T7promoter and terminator, according to the manufacturer's instructions.Reversed-phase high pressure liquid chromatography (HPLC) was carriedout on a GBC HPLC system using a YMC ODS 18 25 mm×25 cm column, elutingwith a gradient of 20%-40% CH₃CN in aqueous TFA over 45 minutes, with aflow rate of 6 ml/min. Gel filtration was carried out on a AKTA FPLCusing a Superdex75 10/30 analytical gel filtration column(Amersham-Pharmacia Biotech), equilibrated and eluted with 20 mM NaPO₄,pH 6.4, at a flow rate of 0.5 ml/min.

Example 1

This example demonstrates the production of an obligate domain-swappeddimer of CVN.

CVN has a pseudosymmetrical, three-dimensional structure comprising twoadjacent triple-stranded anti-parallel β-sheets in the back of theprotein and two oppositely placed β-hairpins on the front of theprotein, each of which is preceded by a single 310 helical turn (Bewleyet al., Nature Struct. Biol. 5: 571-578 (1998)). The homologous sequencerepeats (residues 1-50 and 51-101) are separated by a central linker(comprising Gln50-Pro51-Ser52-Asn53) that precisely crosses overβ-strand 4 (Bewley et al. (1998), supra) and facilitates domain swapping(Yang et al., J. Mol. Biol. 288: 403-412 (1999)). Since the presence ofproline in hinge linkers correlates with domain-swapping (Bergdoll etal., Structure 5: 391-401 (1997)) and Ser52 and Asn53 participate incarbohydrate binding (Bewley (2001), supra), Gln50 was selected fordeletion from the linker so as to generate an obligate dimer.

The CVN Gln50 deletion mutant (ΔQ50-CVN) was constructed bysite-directed mutagenesis and uniformly labeled ¹⁵N-ΔQ50-CVN wasoverexpressed as described previously (Bewley et al. (1998), supra). Therecombinant protein was purified from a crude cell lysate (50% aqueousCH₃CN) in a single step by reversed-phase HPLC. The presence andrelative abundance of monomeric and dimeric wild-type CVN can be readilyassessed from a ¹H-¹⁵N HSQC single quantum coherence correlationspectrum (HSQC), which shows doubling of 18 resolved signals (Bewley etal., J. Am. Chem. Soc. 122: 6009-6016 (2000)). ¹H-¹⁵N HSQC spectra ofNMR samples of ΔQ50-CVN (10% D₂O) prepared with and without adjustingthe pH (measured pH values of 6.4 and 2.3, respectively) were recorded.Unlike wild-type CVN, which shows the presence of approximately 25%domain-swapped dimer upon dissolution (pH approx. 2.3-3.0), the ¹H-¹⁵NHSQC spectrum of ΔQ50-CVN revealed the presence of a single species,regardless of pH. NMR relaxation measurements were carried out todetermine whether this single species was monomeric or dimeric. At 35°C., samples of ΔQ50-CVN had average ¹H_(N) and ¹⁵N T₂ values (Tjandra etal., J. Biomol. NMR 8: 273-284 (1996)) of 22 ms and 93 ms, respectively,and a rotational correlation time (Clore et al., Biochem. 29: 7387-7401(1990)) τc of 9.7 ns (Table 1), values that are only consistent with adimer of approx. 22 kDa. In addition, equilibrium sedimentationmeasurements for ΔQ50-CVN yielded an average molecular mass of 24 (+0.9)kDa, further confirming that ΔQ50-CVN is an obligate dimer. TABLE 1Relaxation Parameters Protein 1HNT2a 15NT2a τcb ΔQ50-CVN ˜22 ms 93 + 4ms 9.7 ns Dimeric CVN ˜20 ms 95 + 6 ms 9.6 ns Monomeric CVN ˜40 ms 167 + 14 ms 4.5 nsa 1HNT2 and 15NT2 values were measured as described in Tjandra et al.(1996), supra.b τc values were calculated from 15NT1/T2 ratios as described in Cloreet al. (1990), supra.

Example 2

This example demonstrates that an obligate domain-swapped dimer of CVNhas enhanced antiviral activity compared to wild-type CVN.

CVN potently inhibits entry into a cell by HIV (Boyd et al., (1997),supra; Dey et al. (2000), supra; and Bewley et al. (2001), supra). Inorder to determine the comparative efficacy of ΔQ50-CVN, ΔQ50-CVN,dimeric wild-type CVN, and monomeric wild-type CVN (obtained after gelfiltration chromatography) were tested in parallel in a quantitativevaccinia virus-based HIV-1 fusion assay (Nussbaum et al., J. Virol. 68:5411-5422 (1994)). ΔQ50-CVN and dimeric wild-type CVN were more potentinhibitors of HIV-1 fusion than monomeric wild-type CVN. Non-linearleast squares best fitting of the titration data to a two-independentsite model (Bewley et al. (2001), supra) for ΔQ50-CVN, dimeric wild-typeCVN, and monomeric wild-type CVN yielded average KDs of 22 nM, 21 nM and67 nM, respectively, with corresponding IC₅₀ values of 9 nM, 9 nM and 32nM. Thus, an obligate domain-swapped dimer of CVN has enhanced antiviralactivity compared to monomeric wild-type CVN.

All of the references cited herein, including journal articles, patents,patent applications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, variations of the preferred embodiments can be used, and itis intended that the invention can be practiced otherwise than asspecifically described herein. Accordingly; this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the claims.

1-21. (canceled)
 22. A purified or isolated obligate domain-swappeddimer of cyanovirin (CVN).
 23. The purified or isolated obligatedomain-swapped dimer of CVN of claim 22, which is a tetravalentcarbohydrate binding protein.
 24. The purified or isolated obligatedomain-swapped dimer of CVN of claim 23, which is stable at a pH fromabout 2.3 to about 8.0.
 25. The purified or isolated obligatedomain-swapped dimer of CVN of claim 22, which comprises the amino acidsequence of wild-type CVN in which there is at least one mutation in thelinker region.
 26. The purified or isolated obligate domain-swappeddimer of CVN of claim 25, in which the at least one mutation is in theregion from about amino acid position 48 to about amino acid position54.
 27. The purified or isolated obligate domain-swapped dimer of CVN ofclaim 26, in which the glutamine at amino acid position 50 is deleted.28. The purified or isolated obligate domain-swapped dimer of CVN ofclaim 26, in which a proline is inserted in the region from about aminoacid position 50 to about amino acid position
 53. 29. The purified orisolated obligate domain-swapped dimer of CVN of claim 26, in which anamino acid in the region from about amino acid position 50 to aboutamino acid position 53 is substituted with a proline.
 30. An isolated orpurified nucleic acid encoding at least one of the obligatedomain-swapped dimer of CVN of claim 22, optionally in the form of avector.
 31. An isolated or purified nucleic acid encoding at least oneof the obligate domain-swapped dimer of CVN of claim 23, optionally inthe form of a vector.
 32. An isolated or purified nucleic acid encodingat least one of the obligate domain-swapped dimer of CVN of claim 24,optionally in the form of a vector.
 33. An isolated or purified nucleicacid encoding at least one of the obligate domain-swapped dimer of CVNof claim 25, optionally in the form of a vector.
 34. An isolated orpurified nucleic acid encoding at least one of the obligatedomain-swapped dimer of CVN of claim 26, optionally in the form of avector.
 35. An isolated or purified nucleic acid encoding at least oneof the obligate domain-swapped dimer of CVN of claim 27, optionally inthe form of a vector.
 36. An isolated or purified nucleic acid encodingat least one of the obligate domain-swapped dimer of CVN of claim 28,optionally in the form of a vector.
 37. An isolated or purified nucleicacid encoding at least one of the obligate domain-swapped dimer of CVNof claim 29, optionally in the form of a vector.
 38. A host cellcomprising the isolated or purified nucleic acid of claim
 30. 39. A hostcell comprising the isolated or purified nucleic acid of claim
 31. 40. Ahost cell comprising the isolated or purified nucleic acid of claim 32.41. A host cell comprising the isolated or purified nucleic acid ofclaim
 33. 42. A host cell comprising the isolated or purified nucleicacid of claim
 34. 43. A host cell comprising the isolated or purifiednucleic acid of claim
 35. 44. A host cell comprising the isolated orpurified nucleic acid of claim
 36. 45. A host cell comprising theisolated or purified nucleic acid of claim
 37. 46. A compositioncomprising the obligate domain-swapped dimer of CVN of claim 22 and acarrier therefor.
 47. A composition comprising the obligatedomain-swapped dimer of CVN of claim 23 and a carrier therefor.
 48. Acomposition comprising the obligate domain-swapped dimer of CVN of claim24 and a carrier therefor.
 49. A composition comprising the obligatedomain-swapped dimer of CVN of claim 25 and a carrier therefor.
 50. Acomposition comprising the obligate domain-swapped dimer of CVN of claim26 and a carrier therefor.
 51. A composition comprising the obligatedomain-swapped dimer of CVN of claim 27 and a carrier therefor.
 52. Acomposition comprising the obligate domain-swapped dimer of CVN of claim28 and a carrier therefor.
 53. A composition comprising the obligatedomain-swapped dimer of CVN of claim 29 and a carrier therefor.
 54. Acomposition comprising the isolated or purified nucleic acid of claim 30and a carrier therefor.
 55. A composition comprising the isolated orpurified nucleic acid of claim 31 and a carrier therefor.
 56. Acomposition comprising the isolated or purified nucleic acid of claim 32and a carrier therefor.
 57. A composition comprising the isolated orpurified nucleic acid of claim 33 and a carrier therefor.
 58. Acomposition comprising the isolated or purified nucleic acid of claim 34and a carrier therefor.
 59. A composition comprising the isolated orpurified nucleic acid of claim 35 and a carrier therefor.
 60. Acomposition comprising the isolated or purified nucleic acid of claim 36and a carrier therefor.
 61. A composition comprising the isolated orpurified nucleic acid of claim 37 and a carrier therefor.
 62. A methodof inhibiting a viral infection of a mammal, which method comprisesadministering to the mammal an antiviral effective amount of thecomposition of claim 46, whereupon the viral infection of the mammal isinhibited.
 63. The method of claim 62, wherein the mammal is a human andthe viral infection is human immunodeficiency viral infection.
 64. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 47, whereupon the viral infection of the mammalis inhibited.
 65. The method of claim 64, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 66. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 48, whereupon the viral infection of the mammalis inhibited.
 67. The method of claim 65, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 68. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 49, whereupon the viral infection of the mammalis inhibited.
 69. The method of claim 65, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 70. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 50, whereupon the viral infection of the mammalis inhibited.
 71. The method of claim 69, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 72. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 51, whereupon the viral infection of the mammalis inhibited.
 73. The method of claim 71, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 74. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 52, whereupon the viral infection of the mammalis inhibited.
 75. The method of claim 73, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 76. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 53, whereupon the viral infection of the mammalis inhibited.
 77. The method of claim 76, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 78. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 54, whereupon the viral infection of the mammalis inhibited.
 79. The method of claim 78, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 80. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 55, whereupon the viral infection of the mammalis inhibited.
 81. The method of claim 80, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 82. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 56, whereupon the viral infection of the mammalis inhibited.
 83. The method of claim 82, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 84. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 57, whereupon the viral infection of the mammalis inhibited.
 85. The method of claim 84, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 86. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 58, whereupon the viral infection of the mammalis inhibited.
 87. The method of claim 86, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 88. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 59, whereupon the viral infection of the mammalis inhibited.
 89. The method of claim 88, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 90. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 60, whereupon the viral infection of the mammalis inhibited.
 91. The method of claim 90, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 92. Amethod of inhibiting a viral infection of a mammal, which methodcomprises administering to the mammal an antiviral effective amount ofthe composition of claim 61, whereupon the viral infection of the mammalis inhibited.
 93. The method of claim 92, wherein the mammal is a humanand the viral infection is human immunodeficiency viral infection.
 94. Amethod of making an obligate domain-swapped dimer of CVN, which methodcomprises introducing at least one mutation in the linker region ofwild-type CVN, whereupon an obligate domain-swapped dimer of CVN isobtained.
 95. The method of claim 94, in which the at least one mutationis in the region from about amino acid position 48 to about amino acidposition
 54. 96. The method of claim 95, in which the glutamine at aminoacid position 50 is deleted.
 97. The method of claim 95, in which aproline is inserted in the region from about amino acid position 50 toabout amino acid position
 53. 98. The method of claim 95, in which anamino acid in the region from about amino acid position 50 to aboutamino acid position 53 is substituted with a proline.